Fox J, Barthold S, Davisson M, Newcomer C, Quimby F, Smith A, eds.
2006. The Mouse in Biomedical Research, 2nd edition Elsevier
Academic Press, San Diego, CA
Volume 3 - Normative Biology, Husbandry, and Models
Chapter 7 Gnotobiotics, pp. 217-233
QUESTIONS:
1.
True or False. Germ free mice are similar anatomically and physiologically to
conventional mice.
2.
True or False. Altered Schaedler flora (ASF) is a cocktail of 8 defined bacteria,
which is now commonly used to associate germ free mice.
3.
True or False. Chlorine dioxide gas, hydrogen peroxide gas and formaldehyde
gas are used commonly to sterilize isolators.
4.
True or False. Gnotobiotic animals include both germ free and defined flora
animals.
ANSWERS
1.
False
2.
True
3
False
4.
True
SUMMARY
Terminology
Gnotobiotic animals (or gnotobiote) – animal stock or strain derived by aseptic
hysterotomy or hysterectomy, embryo transfer or sterile hatching of eggs and are
continuously maintained using aseptic technique where the microbial status of the animal
is fully defined; includes both germ free and defined flora animals
Germfree (axenic) animal – free of all foreign life forms (e.g., bacteria, viruses, etc) apart
from itself; thought to be hypothetical state because indigenous or heretofore
uncharacterized viruses may be integrated into host genome
Defined flora animal – animals maintained in isolated environment and is intentionally
associated with one or more known life forms, usually microorganisms (aka, associated
animal)
Specific pathogen free (SPF) – animals free from specific pathogens but otherwise have
an undefined flora
Restricted flora – gnotobiote associated with altered Schaedler flora from isolator but is
then moved into a maximum barrier room where it can become colonized with additional
organisms (but remains free of adventitial pathogens); higher level of SPF
Conventional animal – animal reared in a room with an unknown microflora and
unknown disease status
Historical perspectives
Nuttall and Thierfelder are considered pioneers of gnotobiotics and germ free research.
Germ free mice have adapted anatomically and physiologically to life without microbes.
Several features that differ between germ-free mice and mice with indigenous microflora:
 germ free mice have enlarged cecum; cecal volume is 5x that of mice with
conventional microflora; torsion, ischemia and obstruction of enlarged cecum
may occur and lead to death
 bowel motility is significantly reduced in germ free mice
 germfree mice have underdeveloped immune system
 lymph nodes are smaller
 level of circulating leukocytes is reduced
 lower levels of serum immunoglobulins as a result of decreased antigenic
stimulation
 heart, lungs and liver of the germfree mouse are smaller than conventional mice
 cardiac output is 1/3 lower
 blood flow to organs and vascular response to catecholamines is reduced
 germfree females have prolonged diestrus period that reduced frequency of estrus
and therefore copulation and implantation rates
Re-colonizing the bowel with commensal or defined microflora can reverse all of these
changes.
A major contribution of gnotobiotic research was the application of technology for the
production and maintenance of disease-free laboratory animals for biomedical research
(via development of isolators, etc practices).
Isolator technology
Isolators are enclosures used to create the sterile environment. They must be made of
material with an impervious physical barrier; main components are the chamber, air
supply, air inlet and outlet, transfer port, and gloves; they come as rigid, semi-rigid, and
flexible film isolators made of plastic or stainless steel. All manipulation of animals and
supplies occurs within the chamber via the use of gloves and sleeves that are attached to
the isolator walls. The glove is the most vulnerable part of the isolator in terms of
contamination potential.
The transfer port is the enclosure that provides a transition between the isolator chamber
and room environment; used for loading and removing items from the chamber; it is the
physical barrier to prevent contamination of the chamber.
Isolators have HEPA filtration on air entry and exhaust. Positive pressure is used to
prevent introduction of airborne contaminants through any punctures and is maintained
when rearing germfree or gnotobiotic animals; if biohazardous agents are used in the
incubator, negative pressure should be used. Air exchange rates are usually higher than
the animal room, some are 30 or more air exchanges per hour inside the chamber. Test
the chamber for leakage using the gas leak detection test.
The long term success of any gnotobiotic operation depends on the sterilization of the
isolator chamber and the equipment/supplies that enter it.
Chemical sterilants
a.
Peracetic acid (PA): first germicidal agent used;
i.
Advantages: high efficacy at low concentrations and low temperatures; is
effective in presence of organic matter; effective in both liquid and vapor
phases
ii.
b.
c.
d.
e.
f.
g.
h.
Other comments: decomposition products are acetic acid, hydrogen
peroxide, oxygen and water; will inactivate the most resistant bacteria and
mold spores but will not penetrate parasite cysts and arthropod eggs; PA
not considered a carcinogen but is shown to be a tumor promoter; highly
corrosive
Chlorine dioxide: most commonly used
i.
Advantages: highly effective; effective in both vapor and liquid phases;
commercially available as Exspor and Clidox;
Hydrogen peroxide and peracetic acid combination (e.g., Spor-Klenz)
i.
Advantages: broad sporicidal efficacy and completely inactivates
Mycobacterium spp. at 20C after 20 minutes contact time;
Formaldehyde gas – carcinogenic, hazardous to use; must be neutralized; not
commonly used
Chlorine dioxide gas – widely used in hospitals; but unlikely for use in
gnotobiotic isolators due to high cost of equipment purchase to provide the gas
Hydrogen peroxide gas –
i.
Advantages: sporicidal activity at low concentrations; non-corrosive; by
products are oxygen and water vapor
ii.
Disadvantages: high initial cost of equipment; poor penetration; requires
fan to help distribute vapor throughout area to be decontaminated
Steam sterilization – Autoclave is most important piece of equipment used to
sterilize food, bedding, water, supplies, etc; high vacuum and pressure models
better than gravity displacement types; verify success of sterilization by using bioindicators (e.g., Geobacillus stearothermophilus preparations);
Irradiation – ionizing radiation of food and bedding has shown to be an
economical and reliable sterilization process; vacuum-sealed radiation-sterilized
packages of food and bedding are commercially available for gnotobiotic
operations;
Ergonomics are critical in the specification and selection of isolators to be used.
Derivation of gnotobiotic mice
Details of the procedure are described briefly in the chapter and elsewhere in more
detail. Briefly, need to have timed germ free females ready to accept the hysterectomy
derived pups from the donor females. The uterus is removed and passed into the isolator
through germicidal dip tank. Once inside the isolator, the uterus is opened and pup
removed, cleaned, and then placed with foster germ free females. Hysterectomy does not
eliminate pathogens that may contaminate fetus after uterine implantation or that are
vertically transmitted (e.g., LCMV, LDH, Pasteurella pneumotropica and Mycoplasma
spp). Vertical transmission of pathogens can be avoided by using embryo transfer
(embryo transfer is another method for germ free production).
Associating germfree mice with defined flora
The organization and equilibrium of the GI ecosystem is defined by the anatomic ad
physiologic characteristics of the GIT and by the tropism of the microbes for those
structures. Most cultivable bacteria in the GIT are facultative anaerobes and obligate
anaerobes (Lactobacillus spp, Enterococcus spp, Enterobacter spp, Bacteroides spp and
Clostridium spp). Obligate anaerobes that represent more than 99% of microflora in GIT
associate with the mucosa throughout the life of the mouse. Lactobacilli were found in
greatest concentration in the stomach, enterococci in the small intestine, and Bacteriodes
and Clostridial spp predominate in the large bowel.
Schaedler defined different ‘cocktails’ of microflora to associate with germ free mice;
these defined flora cocktails reversed some features of germ free mice (e.g., cecal
distention, volvulus incidence, reproductive performance) and made the germ free mice
resistant to infections they were susceptible to by virtue of not having microflora in their
GIT (“colonization resistance”). Antimicrobial administration (e.g., penicillin,
streptomycin) can alter the GI microflora in mice and affect colonization resistance to
opportunistic infections. Opportunistic pathogens are effectively permitted to overgrow
in germ free animals due to the immaturity of the GI tract and naïve immune system.
Studies have shown that the anaerobic fusiform-shaped bacteria in the cecum play a role
in host susceptibility to colonization resistance.
Now there is a standard “altered Schaedler’s flora (ASF) that is used worldwide. This
cocktail includes 8 bacteria (table 7-1, p. 229 in chapter). ASF has been shown to confer
colonization resistance to germ free mice. There are several methods to associate germ
free mice. One is via gavaging pure cultures of living ASF organisms. More commonly,
is inoculating drinking water with fresh feces of cecal contents of mice associated with
ASF organisms.
Monitoring for isolator contamination
A microbiologic monitoring program must be developed to verify germ free status of
mice beginning immediately after rederivation. Briefly, the main principles are
described.
a.
isolator inspection – physically checking isolator and components; olfactory
examination of exhaust air for smell of ammonia (if smells like ammonia, this
suggests that urease-positive bacteria are present and contamination has occurred
since urease positive bacteria are not part of ASF); also testing of animal sentinels
in isolator and work up of any ill or deceased animals
b.
diagnostic procedures –
o microscopic examination of feces for visual assessment of bacterial
morphology present (filamentous, spiral shaped and fusiform shaped
bacteria are present in ASF; any cocci or blunt ended spore forming rods
would indicate contamination)
o microbial culture of feces – need to ensure sterile collection and
appropriate transport media/package so as to prevent entry of oxygen
o microbial culture of various organs from donor animals (i.e., retired
breeders in the isolator)
c.
molecular diagnostics – culture independent methods; e.g., PCR, etc
d.
viral contamination – via serologic assessment of sentinels or donor animals
e.
frequency of monitoring – depends on the risk of contamination and
procedures/access to the incubator;
f.
sources of contamination can include inadequate sterilization of supplies, inadequate
disinfection of glove/sleeve or holes; vertical transmission of pathogens